This invention relates to zibozymes. More particularly, the invention relates to derivatives of wild type hammerhead ribozymes, termed circular, hairpin, circular/hairpin, lariat, and hairpin/lariat hammerhead ribozymes.
Ribozymes are RNA or modified RNA molculecs that can cleave themselves or other nucleic acid molecules (usually RNA) in a catalytic fashion, similar to traditional protein enzymes. The hammerhead ribozyme is one of the smallest ribozymes currently known, and therefore is the most studied of catalytic RNAs. It has shown great utility as a research tool and an antisense therapeutic composition (e.g., U.S. Pat. No. 5,254,678).
The structure and mechanism of the hammerhead ribozyme have been examined using a broad range of approaches. Recently, crystal structures of the hammerhead have been reported. The crystal structures exhibit a Y-shaped configuration for the hammerhead. In this configuration helices I and II form the adjacent upper arms, while helix III forms the lower leg of the Y. Based on these findings, hammerheads in which helix I and II are constrained to remain adjacent and roughly parallel, are expected to be catalytically active. See Sigurdsson, S. T., Tuschl, T., and Eckstein, F. (1995) RNA 1, 575-83.
The metal dependence of folding and cleavage of the hammerhead domain have been examined by several groups using a variety of techniques (Amiri, K. M., and Hagerman, P. J. (1996) J. Mol. Biol. 261, 125-34; Bassi, G. S., Murchie, A. I. H., and lilley, D. M. J. (1996) RNA 2, 756-768; Heus, H. A., and Pardi, A. (1991) J. Mol. Biol. 217, 113-24; Menger, M., Tuschl, T., Eckstein, F., and Porschke, D. (1996) Biochemistry 35, 14710-6; Olita, M., Vinayak, R., Andrus, A., Takagi, Y., Chiba, A., Kaniwa, H., Nishikawa, F., Nishikawa, S., and Taira, K. (1995) Nucleic Acids Symp. Ser. 34, 219-20; Orita, M., Vinayak, R., Andrus, A., Warashina, M., Chiba, A., Kaniwa, H., Nishikawa, F., Nishikawa, S., and Taira, K. (1996) J. Biol. Chem. 271, 9447-54; Simorre, J. P., Iegault, P., Hangar, A. B., Michiels, P., and Pardi, A. (1997) Biochemistry 36, 518-25). These studies indicate that divalent metal ions induce a structural transition, and this coincides with the activation of the hammerhead. Based on these findings, it is believed that an inactive or open conformation is first formed on substrate binding. Subsequently, metal ion addition induces a conformational change to a closed, and catalytically active structure (FIG. 1A). Crystallographic evidence (Scott, W. G., Finch, J. T., and Klug, A. (1995) Cell 81, 991-1002; Scott, W. G., Murray, J. B., Arnold, J.R.P., Stoddard, B. L., and Klug, A. (1996) Science 274, 2065-9; Pley, H. W., Flaherty, K. M., and McKay, D. B. (1994) Nature 372, 68-74), and gel shift (Bassi, G. S., Mollegaard, N. E., Murchie, A. I., von Kitzing, E., and ailley, D. M. (1995) Nat. Struct. Biol. 2,45-55) and fluorescence resonance energy transfer (FRET) analysis (Tuschl, T., Gohlke, C., Jovin, T. M., Westhof, E., and Eckstein, F. (1994) Science 266, 785-9) all support a Y-shaped structure for the closed and active conformation. In this conformation, helices I and II are adjacent and represent the two arms of the Y, while helix III forms the leg (FIG. 1A).
For use of ribozymes in research and especially in therapeutic treatment of various diseases and conditions by antisense-mediated gene inhibition, it would be advantageous to develop hammerhead ribozyme derivatives that have a higher specific activity, have a reduced requirement for magnesium ions, and are more resistant to nuclease degradation than wild-type hammerhead ribozymes.
In view of the foregoing, it will be appreciated that providing hammerhead ribozyme derivatives with these desirable properties would be a significant advancement in the art.